Method for disinfecting and tempering grain

ABSTRACT

Methods for inoculating a grain suitable for use in testing methods of reducing bacterial load are disclosed. Also disclosed are methods of reducing bacterial load on a grain by using elevated temperature and an organic acid. Also disclosed are methods of tempering a grain at an elevated temperature and reduced time as compared to standard tempering methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application represents a National Stage application ofPCT/US2018/029099 originally entitled “Food Safety Method”, filed Apr.24, 2018, pending, which claims the benefit of U.S. ProvisionalApplication Ser. No. 62/489,245, filed Apr. 24, 2017. The entire contentof these applications is incorporated herein by reference.

BACKGROUND

Raw flour can pose a risk of food-borne illness to consumers that eat itraw. While most bacteria remaining on flour cause little, if any,illness in consumers, some virulent bacteria can occasionallycontaminate flour. Shiga toxin producing E. coli (STEC)-associatedillness is mainly associated with undercooked beef, but is occasionallyfound in other agricultural products, such as fresh produce and flour.It is thought that STEC is transferred to crops from the soil directly,or by rain or irrigation splash transfer from the soil, cattle manureused as fertilizer, or water contaminated from cattle-producingfacilities nearby crops. The STEC can adapt to survival on the growingcrops, and contaminate uncooked products, such as flour, produced fromthem.

Although plant parts used to make flour are generally cleaned ofimpurities to reduce risk to human health, there remains somecontamination by bacteria. Whole grain flours are particularlyproblematic, since the surface of the grain is not smooth, and bacteriacan be harbored in the crease of the grain, as well as any othercrevices and cracks on the surface, which are then mixed into the flourupon milling. Bacteria remaining in flour is normally killed when theflour, as a raw ingredient, is cooked. However, raw flour is sometimesmisused by the consumer and is consumed alone or in uncooked foods, suchas uncooked dough.

SUMMARY

The present disclosure relates to methods of treating a grain to reducebacterial load.

Methods of reducing bacterial load on grain are provided herein. Amethod provided herein can include incubating a mixture including thegrain and an organic acid solution for a time and temperature sufficientto provide at least a 3 log reduction in an inoculated grain of the sametype.

In some embodiments, a method of reducing bacterial load on grain caninclude an organic acid solution in the mixture in an amount sufficientto provide an organic acid content of at least about 1.5×10⁻⁵ moles pergram grain and a moisture content to the mixture of from 12% to 20%.

In some embodiments, a method of reducing bacterial load on grain caninclude an organic acid solution having a concentration of organic acidof from 1.5% to 15%.

In some embodiments, a method of reducing bacterial load on grain caninclude a step of preheating the grain prior to incubation with theorganic acid solution.

In some embodiments, a method of reducing bacterial load on grain caninclude incubating for at least 3 hours at a temperature of at least100° F.

In some embodiments, a method of reducing bacterial load on grain caninclude incubating for at least 1 hour at a temperature of from 110° toabout 170° F.

In some embodiments, a method of reducing bacterial load on grain caninclude the use of lactic acid as the organic acid.

In some embodiments, a method of reducing bacterial load on grain can beperformed during tempering of the grain.

Methods of preparing inoculated grain are also provided. A method ofpreparing inoculated grain can include applying a sufficient amount of alog phase inoculation culture to the grain to arrive at a mixture havinga moisture content of from 20% to 30%, agitating the mixturesufficiently to evenly distribute the inoculation culture over thegrain, and drying the agitated mixture at a temperature of from 65° F.to 80° F. for sufficient time to achieve a moisture content of less than15% to produce the inoculated grain. In some embodiments, the dryingstep reduces the moisture content of the inoculated grain to a moisturecontent of less than 12%.

In some embodiments, a log phase inoculation culture used in a method ofpreparing an inoculated grain can have a concentration of from 10⁶ to10¹⁰ CFU/ml.

In some embodiments of a method of preparing an inoculated grain, theagitating step can be performed over a period of from 1 hours to 5hours.

In some embodiments of a method of preparing an inoculated grain, thedrying step can reduce the moisture content of the inoculated grain to amoisture content of less than 12%.

In some embodiments of a method of preparing an inoculated grain, theinoculated grain can have a bacterial load of at least 10⁴ CFU/g grain.

In some embodiments, a log phase inoculation culture used in a method ofpreparing an inoculated grain can contain at least one strain of E.coli.

In some embodiments, a log phase inoculation culture used in a method ofpreparing an inoculated grain can contain at least one Salmonellastrain.

Also provided herein are methods of testing treatments for effect onbacterial load on grain. A method of testing treatments for effect onbacterial load on grain can include providing an inoculated grain,exposing the inoculated grain to treatment to be tested, and enumeratingbacteria on treated grain.

Also provided herein are methods of tempering a grain. A method oftempering grain provided herein can include heating the grain in theabsence of a tempering solution to a temperature of from 115° F. to 150°F., adding sufficient tempering solution to arrive at a moisture contentof 12% to 20% to produce a tempering mixture, and incubating thetempering mixture at 115° F. to 170° F. for sufficient time to achieve atempered grain.

In some embodiments of a method of tempering grain, the temperingsolution includes an organic acid.

In some embodiments of a method of tempering grain, the incubation timeis less than 6 hours.

In some embodiments of a method of tempering grain, the incubation timeis less than 2 hours.

In some embodiments of a method of tempering grain, the grain can bewheat.

Also provided herein are methods of producing a flour. A method ofproducing a flour provided herein can include heating the grain in theabsence of a tempering solution to a temperature of from 115° F. to 150°F., adding sufficient tempering solution to arrive at a moisture contentof 12% to 20% to produce a tempering mixture, incubating the temperingmixture at 115° F. to 170° F. for sufficient time to achieve a temperedgrain, and milling the tempered grain to produce the flour.

In some embodiments of a method of producing a flour, the temperingsolution includes an organic acid.

In some embodiments of a method of producing a flour, the incubationtime is less than 6 hours.

In some embodiments of a method of producing a flour, the incubationtime is less than 2 hours.

In some embodiments of a method of producing a flour, the grain can bewheat.

These and various other features and advantages will be apparent from areading of the following detailed description.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a graph of bacterial load as a function of time and organicacid concentration at 70° F. The dotted line represents the experimental3 log reduction threshold.

FIG. 2 shows a graph of bacterial load as a function of time and organicacid concentration at 100° F. The dotted line represents theexperimental 3 log reduction threshold.

FIG. 3 shows a graph of bacterial load as a function of time and organicacid concentration at 105° F. The dotted line represents theexperimental 3 log reduction threshold.

FIG. 4 shows a graph of bacterial load as a function of time and organicacid concentration at 105° F. The dotted line represents theexperimental 3 log reduction threshold.

FIG. 5 shows a graph of bacterial load as a function of time and organicacid concentration at 110° F. The dotted line represents theexperimental 3 log reduction threshold.

FIG. 6 shows a graph of bacterial load as a function of time and organicacid concentration at 115° F. The dotted line represents theexperimental 3 log reduction threshold.

FIG. 7 shows a graph of bacterial load as a function of time and organicacid concentration at 120° F. The dotted line represents theexperimental 3 log reduction threshold.

FIG. 8 shows a graph of STEC bacterial load as a function of time andorganic acid concentration.

FIG. 9 shows a graph of Salmonella bacterial load as a function of timeand organic acid concentration.

DETAILED DESCRIPTION

Flour is a raw food ingredient made from milling plant parts, such asgrain, that are grown outdoors where bacteria are often present.Although bacteria remaining in flour is normally killed when the flouris cooked, sometimes consumers ingest uncooked flour intentionally orunintentionally. While most consumers may never suffer illness as aresult of consuming raw flour, the occasional illness resulting fromeating raw flour can sometimes be serious. Efforts to further improvesafety of flour were undertaken in order to reduce risk of illness fromconsumption of flour while retaining important functional aspects of theflour.

In an effort to further reduce risk to human health from flour, atechnique for testing survival of a bacterial inoculant on whole grainwas discovered, and is disclosed herein. This discovery made it possibleto experimentally test the effects of treatment on bacterial load ongrain.

It was further discovered, and is disclosed herein, that treatment ofwhole grain with an organic acid and elevated temperature can produce atleast a 3 log reduction in STEC contamination, without significantlychanging the function of the proteins (e.g., gluten) or starch in flourmade with the treated whole grain. This is particularly surprising sinceSTEC are understood to be low pH tolerant and be tolerant of broadtemperature conditions.

An additional discovery disclosed herein, is that the time needed totemper a grain can be reduced to less than 6 hours (e.g., less than 3hours, less than 2 hours, or to 1 hour). Tempering is a method oftreating grain that prepares the grain for milling and helps produceconsistent results when using a milled grain product. Generally,tempering includes adding moisture to a grain before milling. Atempering solution, typically water or water with chlorine (up to about300 ppm), or in the case of a method of reducing bacterial load on agrain provided herein, water with an organic acid, is generally added toa grain and allowed to distribute into the grain. The process canimprove milling characteristics of the grain by altering the bran suchthat it will remain in larger pieces when milled, and making it easierto separate from the endosperm. In addition, tempering can make themoisture content of milled grain products consistent, which allows forgreater predictability in the behavior of milled grain products whenused to make other products, such as flour. Tempering typically takes atleast 6 hours for softer grains, such as soft wheat, to more than 24hours for harder grains, such as hard wheat. It was discovered that, bypreheating a grain prior to the addition of a tempering solution and aheated incubation time, tempering can be done in less than 6 hours, lessthan 2 hours, or 1 hour, or less.

Inoculated Grain

A new method of preparing an inoculated grain is provided herein. Insome embodiments, the method includes applying a sufficient amount of alog phase inoculation culture to a grain (e.g., wheat, oat, barley, orthe like) in order to arrive at a mixture that has a moisture content offrom 20% to 30% (e.g., from about 22% to about 27%, or from about 24% toabout 25%), agitating the mixture sufficiently to evenly distribute theinoculation culture over the grain, and drying the agitated mixture toarrive at an inoculated grain. Moisture content of a grain, grainmixture, or inoculated grain described herein can be measured bygrinding the grain and measuring moisture content using a moistureanalyzer (e.g., Metler Toledo HB43-S moisture analyzer).

In some embodiments, a method of preparing inoculated grain includesapplying a sufficient amount of a log phase inoculation culture to agrain having a moisture content of 5-8% to arrive at a mixture having amoisture content of from 9% to 13%, and agitating the mixturesufficiently to evenly distribute the inoculation culture over the grainto arrive at an inoculated grain. Moisture content of a grain, grainmixture, or inoculated grain described herein can be measured bygrinding the grain and measuring moisture content using a moistureanalyzer (e.g., Metier Toledo HB43-S moisture analyzer).

An inoculated grain provided herein can have a stable bacterial loadduring storage. For example, an inoculated grain provided herein canhave a bacterial load varies by no more than 3 log over a storage timeof at least 30 days at 70° F. in a sealed container. In someembodiments, an inoculated grain provided herein can have a bacterialload that does not fall below a desired threshold (e.g., 10⁵ CFU/g grainor 10⁴ CFU/g grain) over a storage time of at least 30 days at 70° F. ina sealed container.

As used herein, a “log phase inoculation culture” is a liquid culturecontaining one or more strains of logarithmically growing bacteria. Alog phase inoculation culture includes any appropriate bacterial speciesfor testing bacterial survival on grain. Although virulent strains ofbacteria can be used, non-virulent representative strains may bepreferable. For example, if STEC bacteria are to be used, attenuatedstrains can be used instead of virulent strains of STEC, such as O121,O157:H7, O26:H4, O103:K:H8, and O111a, 111b:K58:H21. In another example,if Salmonella bacteria are to be used, strains such as American TypeCulture Collection (ATCC, Manassas, Va., USA) accessions ATCC #51741,ATCC #51957, ATCC #BAA-707, ATCC #BAA-2568, and ATCC #9268 can be used.

A log phase inoculation culture can be made using any appropriatetechnique. An example includes inoculating a liquid bacterial medium(e.g., brain heart infusion medium, tryptic soy broth, and/or otherselective or non-selective broth) with a colony from a solid bacterialmedium (e.g., MacConkey agar, sorbitol-MacConkey agar, plate count agar,and/or other selective or non-selective medium), and allowing thebacteria to grow in the liquid medium in appropriate conditions (e.g.,incubation at about 37° C.) until logarithmic phase growth is reached.In some embodiments, a liquid medium can be inoculated using bacteriafrom a liquid culture. Methods for inoculating and incubating a liquidculture can be combined to arrive at a desired log phase inoculationculture.

In some embodiments, multiple bacterial strains can be grown together ina liquid culture to reach log phase. However, in order to reducecompetition of bacteria in mixed cultures, in some embodiments,different bacterial strains can be grown separately to log phase beforecombining into a single log phase inoculation culture.

A log phase inoculation culture can contain from about 10⁶ to about 10¹⁰(e.g., from about 10⁷ to about 10⁹, or about 10⁸) colony forming units(CFU) per ml of liquid culture. Colony forming units per ml can bedetermined using any appropriate method, such as by measuring opticaldensity of the culture or by counting cells by flow cytometry.

The amount and concentration of a log phase inoculation culture appliedto grain can be adjusted as desired to result in an inoculated grainthat has the desired bacterial load. A suitable bacterial load of aninoculated grain provided herein can be at least 10³ CFU/g grain (e.g.,at least 10⁴ CFU/g grain, or from 10⁵ to 10⁹ CFU/g grain, or from about10⁵ to about 10⁷ CFU/g grain). Bacterial load on an inoculated grain canbe enumerated using any appropriate method. For example, bacterial loadcan be measured by adding grain to a dilution buffer and agitating themixture. The buffer can then be serially diluted (e.g., dilutions from10⁻¹ to 10⁻⁶) and plated on to an agar plate or film. Agar plates orfilms can then be incubated at a temperature appropriate for thebacteria to be enumerated (e.g., 32-37° C. for STEC bacteria), andbacterial colonies enumerated after an appropriate incubation period(e.g., 18-24 hours).

In a method of preparing an inoculated grain provided herein, followingapplication of a log phase inoculation culture to a grain, the mixturecan be agitated using any appropriate means (e.g., stirring, shaking,vibration, tumbling, or the like) for sufficient time to evenlydistribute the log phase inoculation culture throughout the grain. Forexample, a log phase inoculation culture and a grain can be combined ina container, which is then shaken periodically over several hours toevenly distribute the log phase inoculation culture throughout thegrain.

A method of preparing an inoculated grain further includes drying amixture of log phase inoculation culture and grain that has beenagitated to evenly distribute the inoculation culture throughout thegrain. Drying can be done at a temperature of from 60° F. to 85° F.(e.g., 65° F. to 80° F.) for sufficient time to achieve an inoculatedgrain having a moisture content of less than 15% (e.g., less than 13%,or less than 12%).

Any appropriate drying method can be used to produce an inoculatedgrain. For example, a mixture of log phase inoculation culture and grainthat has been agitated to evenly distribute the inoculation culturethroughout the grain can be spread out on trays and dried at ambientconditions. In another example, a mixture of log phase inoculationculture and grain that has been agitated to evenly distribute theinoculation culture throughout the grain can be dried in an oven. Insome embodiments, drying can be done using multiple methods and/or inmultiple stages. For example, a mixture can be dried in ambientconditions for some period of time, followed by drying in an oven ordrying by evaporation (e.g., by placing in a ventilated hood).

In some embodiments, an inoculated grain provided herein retainssubstantially all of the structural and functional characteristics ofthe grain before inoculation. For example, an inoculated grain canretain substantially the same gross structural characteristics, as wellas substantially the same protein content and functionality, and starchcontent and functionality, as before inoculation.

Methods of Reducing Bacterial Load

An inoculated grain provided herein can be used to test the effect of atreatment on bacterial load of a grain. A method for testing a treatmentfor its effect on bacterial load on grain includes exposing aninoculated grain to the treatment to be tested, and enumerating bacteriaon the grain as described above.

It was discovered that, by using an inoculated grain provided herein,the effect of an organic acid on bacterial load, as well as incubationtemperature could be tested. It was further discovered that acombination of an organic acid (e.g., lactic acid, citric acid, orsalicylic acid) and an incubation time at a range of temperatures can beused to reduce bacterial load on a grain. It is believed that a methodof reducing a bacterial load on a grain provided herein can be used toreduce the risk of bacterial contamination of grain prior to millingand/or use of the grain in foods. In some embodiments, a method ofreducing bacterial load on grain provided herein can be used during, orin addition to, tempering of the grain (e.g., using American Associationof Cereal Chemists (AACC) method 26-95.01).

In some embodiments, a method of reducing bacterial load on a grainprovided herein can be sufficient to reduce bacterial load by at least 3log (e.g., at least 4 log, or at least 5 log) on an inoculated grain. Agrain treated using a method of reducing bacterial load provided hereinneed not actually experience a 3 log reduction in bacterial load, solong as the treatment is sufficient to do so on an inoculated grainprovided herein.

In some embodiments, a method of reducing bacterial load on a grain caninclude adding an organic acid solution to the grain in an amountsufficient to provide a mixture having at least 1.5×10⁻⁵ (e.g., 1.5×10⁻⁵to 1×10⁻⁴) moles acid per gram grain, and a moisture content of fromabout 12% to about 20% (e.g., from about 14% to about 18%). An organicacid solution used in a method provided herein can include anyappropriate concentration of an organic acid, such as lactic acid,peracetic acid, acetic acid, citric acid, or salicylic acid. Forexample, a 1.5% to 10% (e.g., about 2% to about 8%) lactic acid solutioncan be used in a method provided herein. Since the amount of moistureadded to a grain can vary depending on the initial moisture content ofthe grain and the desired moisture content once the grain is tempered,an amount of organic acid can be added as an amount by weight of thegrain treated. For example, an organic acid, such as lactic acid, can beadded in an amount of 0.6 g to 2 g (e.g., from about 0.625 g/to 1.875 g)per pound of grain.

In some embodiments, a method of reducing bacterial load on a grainincludes incubating a mixture of an organic acid solution and grain at atemperature between 70° F. and 170° F. (e.g., from about 120° F. toabout 150° F., or from about 120° F. to about 140° F.) for sufficienttime to reduce the bacterial load by at least 2 log (e.g., at least 3log, at least 4 log, or at least 5 log). It is to be understood that ahigher temperature and/or higher organic acid concentration can reducethe time sufficient to reduce the bacterial load. Conversely, a longertime combined with a lower temperature and/or lower organic acidconcentration can be sufficient to achieve a 3 log reduction. Lacticacid is particularly effective in a method of reducing bacterial load ona grain, however it is to be understood that a higher temperature and/orhigher organic acid concentration can be used to supplement a methodusing a less effective organic acid. Preferably, the combination oftime, temperature, and acid content used in a method of reducingbacterial load does not significantly affect the functionality of thegrain following treatment. For example, a flour made from a treatedgrain should function in substantially the same way as a grain that hasnot been treated.

A method of reducing bacterial load on a grain need not be a continuousprocess, and the incubation temperature need not be constant. Forexample, a total exposure time and temperature at a particular organicacid concentration can be calculated as being sufficient to reducebacterial load by at least 3 log, and so long as the total exposuretime, temperature and organic acid concentration are met, the treatmentcan still be considered sufficient to reduce the bacterial load by atleast 3 log. This can be particularly useful if a method of reducingbacterial load is performed as part of a tempering treatment of thegrain, since tempering times and temperatures can vary during thetempering process.

In some embodiments, a method of reducing bacterial load provided hereinmay be desired to result in reducing bacterial load by at least 3 logwithin 4 hours of application of an organic acid. In some embodiments, amethod of reducing bacterial load provided herein can take from 4-24hours (e.g., 8-24 hours) to be sufficient to reduce the bacterial loadby at least 3 log. In some embodiments, a 3 log reduction can beachieved within 1 hour of treatment (e.g., at about 125° F. to about140° F. with from about 0.6 g to about 2 g lactic acid per pound ofgrain).

Any appropriate equipment can be used in a method of reducing bacterialload on a grain. For example, grain storage, mixing, and heatingequipment can be used. In some embodiments, in order to reduce damage toequipment used in a method of reducing bacterial load, equipment thatcomes in contact with an organic acid can have contact surfaces thatinclude materials that are resistant to acid damage, such as stainlesssteel.

Accelerated Grain Tempering

Methods of accelerating tempering a grain are also provided. A methodfor tempering a grain includes preheating the grain prior to addition ofa tempering solution. A grain can be preheated to a temperature of atleast 115° F. (e.g., from 115° F. to about 170° F., from about 120° F.to about 150° F., or from about 120° F. to about 140° F.). In someembodiments, a preheating step is performed on grain having a moisturecontent of from 10% to 12%. Preheating a grain can be followed byaddition of a tempering solution immediately upon the grain reaching anaverage temperature of at least 115° F. (e.g., about 115° F. to about170° F., from about 120° F. to about 140° F., or about 125° F.), or thegrain can be held at the preheat temperature for any appropriate time(e.g., several minutes up to several hours) before the addition of atempering solution.

A tempering solution, preheated to about the temperature of thepreheated grain to which it will be added, can be added to a preheatedgrain in an amount sufficient to arrive at a moisture content of thegrain of about 12% to about 20% (e.g., about 14% to about 18%, or about15%) to produce a tempering mixture. Any suitable tempering solution canbe used. However, if it is also desired to reduce bacterial load of thegrain, a tempering solution that includes an organic acid describedabove, or a tempering solution containing an amount of chlorine (e.g.,200 ppm to 300 ppm chlorine), can be used.

A tempering mixture can be incubated at temperature sufficient toproduce a tempered grain within 6 hours (e.g., within 3 hours, within 2hours, or within 1 hour) of producing the tempering mixture. In someembodiments, such as where a tempering solution is used that contains anorganic acid, the incubation time and temperature can be sufficient toprovide at least a 3 log reduction in an inoculated grain of the sametype. The temperature used during the tempering step of an acceleratedtempering method provided herein can be the same temperature or adifferent temperature (i.e., higher or lower) than the preheatedtemperature of the grain.

In some embodiments, a method of tempering a grain provided herein canalso include a step of reducing the temperature of the tempered grain toa temperature below 115° F. (e.g., below 100° F. or below 80° F.).

A tempered grain produced using an accelerated tempering proceduredescribed herein can be used in the same way as a grain that has beentempered using a standard procedure, such as that described in AmericanAssociation of Cereal Chemists (AACC) method 26-95.01. For example, agrain tempered in a method provided herein can be milled to produce aflour.

The following examples illustrate particular embodiments and are notintended to limit the claimed invention.

EXAMPLES Example 1 Log Phase Inoculation Culture

Five strains of attenuated STEC shown in Table 1 were obtained from theAmerican Type Culture Collection (ATCC®, Manassas, Va., USA) and used toinoculate 10 ml of brain heart infusion (BHI) broth to produce 5separate starter cultures.

TABLE 1 ATCC No. Serotype BAA-2212 E. coli O26:H4 23982 E. coliO103:K:H8 29552 E. coli O111a, 111b:K58:H21 BAA-2190 E. coli O121 43888E. coli O157:H7

The starter cultures were incubated at 37° C. for 24 hours to reach logphase growth. The log phase starter cultures were each streaked ontoseparate CHROMager™ STEC selective agar plates (CHROMagar™, Paris,France), and incubated at 37° C. for 18-24 hours. Isolated colonies fromthe STEC selective agar were then streaked onto individual plate countagar (PCA) plates, and incubated at 37° C. for 24 hours. Isolatedcolonies from the PCA plates were used to inoculate 5 separate samplesof 10 ml of BHI broth, which were incubated at 37° C. for 24 hours tolog phase growth and an approximate concentration of 10⁹ CFU/ml. 100 μlof each of the BHI broths was used to inoculate 1 L of BHI brothcultures, which were incubated at 37° C. for 24 hours to log phasegrowth and an approximate concentration of 10⁸ CFU/ml. The 5 1 L BHIcultures were combined to produce a log phase inoculation culture at aconcentration of 1.3×10⁸ CFU/ml.

Example 2 Grain Inoculation

The log phase inoculation culture from Example 1 was added to hard redwinter (HRW) wheat with a 10% moisture content in a ratio of 192 g logphase inoculation culture to 1 kg wheat to arrive at a moisture contentof about 24.6%. The mixture was sealed in a container and shakenvigorously every 30 minutes over 3 hours at room temperature. The wheatwas then spread out onto baking trays, and the wheat was allowed to dryat ambient lab conditions (about 70° F. and about 34.5% relativehumidity) for 3-5 days prior to double bagging into plastic bags forstorage at ambient lab temperature. The moisture content of the wheat atbagging was about 13.8%, and the bacterial load was approximately1.3×10⁸ CFU/g. Bacteria on the inoculated wheat was enumeratedperiodically over 30 days. After an initial drop of 1-2 log, the E. colipopulation stabilized on the wheat after 15 days at about 10⁶ CFU/g.Enumeration of bacterial load was performed by adding 11 g wheat to 99ml dilution buffer, then agitating in a stomacher. The buffer was thenserially diluted from 10⁻¹ to 10⁻⁶ and plated onto a petri film. Thepetri film was incubated at 32-37° C. and bacterial colonies wereenumerated after 24 hours.

To measure the effect of drying on bacterial load, HRW wheat wasinoculated as above, but instead of bagging after drying at ambientconditions, the samples were further dried at 70° F. and 20% relativehumidity, and sampled for moisture content at 30 minute intervals over a3 hour period of time in order to more closely approximate the moisturecontent of wheat typically used for commercial milling. These dryingconditions approximate conditions typically encountered at a grain mill.While the moisture content of the wheat was reduced at a rate of about0.47% moisture per hour, the bacterial load was steady between 10⁵/g and10⁶/g, indicating that further drying had no significant effect onpathogen population on the inoculated wheat.

Based on these results, it appears as though a grain can be inoculatedwith STEC bacteria and dried to produce a grain that is stablyinoculated with dry-adapted STEC bacteria. It is believed that aninoculated grain can be used as a model for dry-adapted bacteria,particularly STEC, naturally occurring on crops.

Also based on these results, it appears that the initial inoculationrate should be 1-2 log higher than the final desired bacterial load. Afinal bacterial load of from about 10⁴ to 10⁷ should be sufficient toprovide statistical confidence in the effects of tested treatmentconditions on bacterial load, though the level of initial bacterial loadmay be adjusted as desired.

Similar results were obtained using a blend of Salmonella strains,including ATCC accessions ATCC #51741, ATCC #51957, ATCC #BAA-707, ATCC#BAA-2568, and ATCC #9268. These results indicate that inoculated graincan be produced using STEC bacteria, as well as other bacteria types,such as Salmonella.

Example 3 Effect of Temperature and Organic Acid on Bacterial Load

HRW wheat was inoculated with a 5 strain STEC cocktail, as described inExample 1. The drying procedure was modified to replace the 5 day dryingat ambient conditions with 12-24 hours in the sealed container at about70° F., followed by drying for 3 days at ambient conditions on traysprior to bagging and storage at ambient temperature. The inoculatedwheat had a moisture content of 14-15% and bacterial load of about 10⁶CFU/g.

Samples for testing included 250 g of inoculated wheat in plasticNalgene containers. The amount of organic acid solution added to eachsample was calculated to be 11.78 g of solution per 250 g inoculatedwheat to mimic tempering moisture conditions (15% moisture) according toAmerican Association of Cereal Chemists (AACC) method 26-95.01. Theorganic acid concentrations in % (tempering solution), organic acidconcentrations in moles/g wheat, incubation temperatures, and startingbacterial load are provided in Table 2.

TABLE 2 Moles acid/g Temperature Starting Treatment Tempering solutionwheat (° F) CFU/g 1 water 0 70 2.8 × 10⁶ 2 water 0 105 4.7 × 10⁵ 3 3.85%lactic acid 2.02 × 10⁻⁵ 70 2.8 × 10⁶ 4 3.85% lactic acid 2.02 × 10⁻⁵ 1054.7 × 10⁵ 5 7.77% lactic acid 4.03 × 10⁻⁵ 70 2.8 × 10⁶ 6 7.77% lacticacid 4.03 × 10⁻⁵ 105 4.7 × 10⁵ 7 11.55% lactic acid 6.06 × 10⁻⁵ 70 2.8 ×10⁶ 8 11.55% lactic acid 6.06 × 10⁻⁵ 105 4.7 × 10⁵ 9 15.4% lactic acid8.08 × 10⁻⁵ 70 2.8 × 10⁶ 10 15.4% lactic acid 8.08 × 10⁻⁵ 105 4.7 × 10⁵11 water 0 105 2.1 × 10⁶ 12 water 0 120 2.1 × 10⁶ 13 3.85% lactic acid2.02 × 10⁻⁵ 105 2.1 × 10⁶ 14 3.85% lactic acid 2.02 × 10⁻⁵ 120 2.1 × 10⁶15 5.78% lactic acid 3.01 × 10⁻⁵ 105 2.1 × 10⁶ 16 5.78% lactic acid 3.01× 10⁻⁵ 120 2.1 × 10⁶ 17 7.77% lactic acid 4.03 × 10⁻⁵ 105 2.1 × 10⁶ 187.77% lactic acid 4.03 × 10⁻⁵ 120 2.1 × 10⁶ 19 11.55% lactic acid 6.06 ×10⁻⁵ 105 2.1 × 10⁶ 20 11.55% lactic acid 6.06 × 10⁻⁵ 120 2.1 × 10⁶ 21300 ppm chlorine 0 105 2.1 × 10⁶ 22 water 0 100 3.4 × 10⁶ 23 water 0 1153.4 × 10⁶ 24 3.85% lactic acid 2.02 × 10⁻⁵ 100 3.4 × 10⁶ 25 3.85% lacticacid 2.02 × 10⁻⁵ 110 3.4 × 10⁶ 26 3.85% lactic acid 2.02 × 10⁻⁵ 115 3.4× 10⁶ 27 5.78% lactic acid 3.01 × 10⁻⁵ 100 3.4 × 10⁶ 28 5.78% lacticacid 3.01 × 10⁻⁵ 110 3.4 × 10⁶ 29 5.78% lactic acid 3.01 × 10⁻⁵ 115 3.4× 10⁶ 30 7.77% lactic acid 4.03 × 10⁻⁵ 100 3.4 × 10⁶ 31 7.77% lacticacid 4.03 × 10⁻⁵ 110 3.4 × 10⁶ 32 7.77% lactic acid 4.03 × 10⁻⁵ 115 3.4× 10⁶

Inoculated wheat samples were incubated for up to 24 hours, and thebacterial loads were measured various time points as shown in Tables 3-9and graphed in FIGS. 1-7 .

TABLE 3 Treatment at 70° F Treatment Time 1 3 5 7 9 (hr) CFU/g wheat 02.8 × 10⁶ 2.8 × 10⁶ 2.8 × 10⁶ 2.8 × 10⁶ 2.8 × 10⁶ 4 2.0 × 10⁶ 2.0 × 10⁵6.2 × 10⁴ 4.6 × 10⁴ 7.7 × 10³ 8 1.3 × 10⁶ 6.2 × 10⁵ 5.6 × 10⁴ 4.8 × 10³3.8 × 10³ 12 1.9 × 10⁶ 3.7 × 10⁵ 5.7 × 10⁴ 2.1 × 10⁴ 6.0 × 10³ 24 1.3 ×10⁶ 2.9 × 10⁵ 3.2 × 10⁴ 1.0 × 10⁴ 1.0 × 10³

TABLE 4 Treatment at 105° F Treatment Time 2 4 6 8 10 (hr) CFU/g wheat 04.7 × 10⁵ 4.7 × 10⁵ 4.7 × 10⁵ 4.7 × 10⁵ 4.7 × 10⁵ 4 1.1 × 10⁵ 1.7 × 10⁴4.4 × 10³ 2.5 × 10² 6.0 × 10¹ 8 4.7 × 10⁴ 1.8 × 10⁴ 3.0 × 10² 7.0 × 10¹4.0 × 10¹ 12 3.0 × 10⁴ 2.4 × 10³ 2.3 × 10² 1.8 × 10³ 3.0 × 10¹ 24 6.5 ×10³ 4.2 × 10² 1.1 × 10² 2.0 × 10¹ 2.0 × 10¹

TABLE 5 Treatment at 105° F. Treatment Time 11 13 15 17 19 21 (hr) CFU/gwheat 0 2.1 × 10⁶ 2.1 × 10⁶ 2.1 × 10⁶ 2.1 × 10⁶ 2.1 × 10⁶ 2.1 × 10⁶ 42.1 × 10⁵ 3.9 × 10⁴ 8.3 × 10³ 4.9 × 10³ 2.4 × 10² 3.8 × 10⁵ 8 1.7 × 10⁵3.0 × 10⁴ 2.0 × 10⁴ 3.1 × 10³ 4.3 × 10² 2.2 × 10⁵ 12 4.4 × 10⁴ 5.2 × 10³2.6 × 10³ 9.6 × 10² 2.0 × 10¹ 1.8 × 10⁴ 24 4.9 × 10⁴ 8.7 × 10² 7.0 × 10²5.8 × 10² 4.0 × 10¹ 1.0 × 10⁴

TABLE 6 Treatment at 120° F Treatment Time 12 14 16 18 20 (hr) CFU/gwheat 0 2.1 × 10⁶ 2.1 × 10⁶ 2.1 × 10⁶ 2.1 × 10⁶ 2.1 × 10⁶ 4 1.5 × 10⁴7.1 × 10² 4.0 × 10² 2.0 × 10¹ NM* 8 8.9 × 10³ 5.1 × 10² 1.3 × 10² 2.0 ×10¹ NM  12 5.8 × 10² 4.0 × 10¹ 2.0 × 10¹ 1 2.0 × 10¹ 24 5.0 × 10¹ 1 1 11 *NM = not measured

TABLE 7 Treatment at 100° F Treatment Time 22 24 27 30 (hr) CFU/g wheat0 3.4 × 10⁶ 3.4 × 10⁶ 3.4 × 10⁶ 3.4 × 10⁶ 4 3.6 × 10⁶ 1.7 × 10⁵ 7.7 ×10⁴ 1.6 × 10⁴ 8 2.1 × 10⁶ 2.3 × 10⁵ 4.5 × 10⁴ 1.1 × 10⁴ 12 5.7 × 10⁵ 5.3× 10⁴ 3.1 × 10⁴ 3.9 × 10³ 24 6.0 × 10⁵ 3.3 × 10⁴ 7.5 × 10³ 1.5 × 10³

TABLE 8 Treatment at 110° F Treatment Time 25 28 31 (hr) CFU/g wheat 03.4 × 10⁶ 3.4 × 10⁶ 3.4 × 10⁶ 4 2.1 × 10⁶ 6.5 × 10³ 3.6 × 10³ 8 1.6 ×10⁴ 8.0 × 10³ 2.0 × 10³ 12 1.5 × 10³ 5.3 × 10³ 4.8 × 10² 24 8.1 × 10²3.6 × 10³ 8.5 × 10¹

TABLE 9 Treatment at 115° F Treatment Time 23 26 29 32 (hr) CFU/g wheat0 3.4 × 10⁶ 3.4 × 10⁶ 3.4 × 10⁶ 3.4 × 10⁶ 4 2.3 × 10⁵ 2.1 × 10⁴ 2.7 ×10³ 1.4 × 10³ 8 2.9 × 10⁵ 6.1 × 10⁴ 2.0 × 10³ 5.2 × 10² 12 4.8 × 10⁴ 2.1× 10³ 3.7 × 10² 8.0 × 10¹ 24 4.7 × 10⁴ 1.4 × 10² 5 5

As can be seen in Tables 3-6 and FIGS. 1-7 , a three log reduction wasachieved with several of the treatments. Table 10 shows the treatmentsthat achieved a 3 log reduction within 4 hours. Table 11 shows thetreatments that achieved a 3 log reduction within 8 hours. Table 12shows the treatments that achieved a 3 log reduction within 12 hours.Table 13 shows the treatments that achieved a 3 log reduction within 24hours.

TABLE 10 3 log reduction within 4 hours Incubation % lactic acid Molesacid/g wheat temperature (° F.) 11.55% lactic acid 6.06 × 10⁻⁵ 105 15.4%lactic acid 8.08 × 10⁻⁵ 105 5.78% lactic acid 3.01 × 10⁻⁵ 115 7.7%lactic acid 4.03 × 10⁻⁵ 115 3.85% lactic acid 2.02 × 10⁻⁵ 120 5.78%lactic acid 3.01 × 10⁻⁵ 120 7.7% lactic acid 4.03 × 10⁻⁵ 120 11.55%lactic acid 6.06 × 10⁻⁵ 120

TABLE 11 -3 log reduction within 8 hours Incubation % lactic acid Molesacid/g wheat temperature (° F.) 7.7% lactic acid 4.03 × 10⁻⁵ 110 5.78%lactic acid 3.01 × 10⁻⁵ 115

TABLE 12 -3 log reduction within 12 hours Incubation % lactic acid Molesacid/g wheat temperature (° F.) 7.7% lactic acid 4.03 × 10⁻⁵ 105 3.85%lactic acid 2.02 × 10⁻⁵ 115 water 0 120

TABLE 13 -3 log reduction within 24 hours Incubation % lactic acid Molesacid/g wheat temperature (° F.) 15.4% lactic acid 8.08 × 10⁻⁵ 70 7.7%lactic acid 4.03 × 10⁻⁵ 100 3.85% lactic acid 2.02 × 10⁻⁵ 105 5.78%lactic acid 3.01 × 10⁻⁵ 105 3.85% lactic acid 2.02 × 10⁻⁵ 110

Example 4 Accelerated Tempering

While performing testing described in Example 3, above, it was notedthat a sample had experienced a drop in water activity within an hour ofbeginning incubation. Since a water activity drop can be an indicator oftempering quality, further experimentation was performed to determinewhether tempering time could be accelerated.

In order to test the efficacy of accelerated tempering, wheat wastreated with a preheat step followed by a 1 hour temper step at elevatedtemperature. Briefly, ambient temperature wheat was heated to atemperature of 125° F.±5° F. without the addition of a temperingsolution. The preheated wheat was then transferred to a steam jacketedkettle and a lactic acid solution as a tempering solution was added at atemperature of 120° F.±10° F. to achieve a 15.5% wetted wheat moistureand a lactic acid concentration of 1.2 g per lb wheat (2.94×10⁻⁵ molesper gram wheat). The wheat and tempering solution was mixed at slowspeed for 1 minute and transferred to a poly-lined 5 gallon pail andincubated at 125° F. A button temperature probe in the geometric centerof the wheat in the pail was used to monitor temperature of the wheat.After an hour, the wheat was cooled in a glycol jacketed mixer to lessthan 80° F. The tempered wheat was milled either immediately aftercooling or allowed to rest for 18 hours at ambient temperature followingcooling. Milling performance and flour attributes of the tempered wheat,including attributes of dough made with the flour, were tested andcompared to a control wheat sample that was treated identically, exceptthat the wheat was not preheated, and the tempering was performed atambient conditions overnight instead of heated conditions for 1 hour.The results of the milling performance and flour attributes are shown inTable 14.

TABLE 14 Milling performance and flour attributes One-hour heated temper18 hour ambient Attribute Control No ambient hold hold Starting wheat10.53 10.53 10.53 moisture (%) Starting wheat bulk 828 828 828 density(kg/m³) Bulk density 750 780 782 tempered wheat prior to milling (kg/m³)Moisture tempered 15.66 14.81 Not measured wheat prior to milling (%)Milling yield (%) 70.5 70.3 70.4 Flour moisture (%) 14.15 13.39 13.65Flour moisture - 798 780 769 corrected BU (absorption) Flour stability20.8 17.3 19.2 (W*hr/kg) Flour-extensibility 83 81 81 (mm) Flour-Rmax(BU) 749 734 748 Flour-Area under 88 84 87 curve (BU*mm)

As can be seen in Table 14, the wheat tempered for 1 hour, and flourmade therefrom, performed similarly to wheat tempered at ambientconditions overnight, and flour made therefrom.

In order to determine whether accelerated temper conditions could beused to reduce bacterial load on a grain, inoculated grain with eitherSTEC or Salmonella described in Example 1 was treated with a preheatstep to 110° F., 120° F., and 130° F. and treated with temperingsolutions containing 3.85% or 5.78% lactic acid, and the bacterial loadwas measured at timed intervals. Tables 15 and 16, graphed in FIGS. 8and 9 , respectively, show the results of the bacterial load over time.

TABLE 15 Bacterial load-STEC Temperature 110° F. 120° F. 130° F. Lacticacid concentration 3.85% 5.78% 3.85% 5.78% 3.85% 5.78% Time BacterialLoad 0  1.1 × 10⁷  1.2 × 10⁷ 4.5 × 10⁶ 1.7 × 10⁶ 6.4 × 10⁶ 6.8 × 10⁶ 18.65 × 10⁴  1.18 × 10⁴**  1.05 × 10³**  8.65 × 10²**  3.18 × 10³**  7.5× 10^(1†) 2   1.5 × 10⁴** 4.55 × 10³ 4.4 × 10³ 3.1 × 10³ 3.4 × 10² 5.0 ×10¹ 3 1.62 × 10⁴ 3.55 × 10³ 1.31 × 10³  6.7 × 10² 1.5 × 10² 1.2 × 10² 41.75 × 10⁴  2.8 × 10³ 2.4 × 10³ 3.75 × 10²  1.55 × 10²  3.0 × 10¹ 8 2.05× 10⁴ 1.03 × 10³ 2.75 × 10²  8.0 × 10¹ 1^(†) 1 **= 3 log reduction ^(†)=5 log reduction

TABLE 16 Salmonella Temperature 110° F. 120° F. 130° F. Lactic acidconcentration 3.85% 5.78% 3.85% 5.78% 3.85% 5.78% Time Bacterial Load 08.7 × 10⁶ 3.3 × 10⁷ 5.0 × 10⁶ 4.0 × 10⁶ 4.3 × 10⁶ 6.1 × 10⁶ 1 1.4 × 10⁵6.5 × 10⁴ 2.7 × 10⁴ 8.2 × 10⁴ 7.0 × 10⁴  4.25 × 10²** 2 2.95 × 10⁵  4.65× 10⁴  2.95 × 10³  4.3 × 10⁴ 2.0 × 10⁴ 3.4 × 10² 3 9.5 × 10⁴  2.8 ×10⁴**  5.67 × 10⁴** 3.2 × 10⁴  3.85 × 10³** 7.0 × 10¹ 4 9.2 × 10⁵ 9.5 ×10³ 2.3 × 10³ 1.57 × 10⁴  2.55 × 10³  8.0 × 10¹ 8 1.7 × 10⁴ 9.5 × 10²7.45 × 10²   3.55 × 10³** 1.9 × 10³  2.5 × 10^(1†) **= 3 log reduction^(†)= 5 log reduction

As can be seen in Table 15, STEC bacterial load can be reduced by atleast 3 log within 1 to 2 hours (e.g., within 40 minutes) by preheatinga grain followed by a heated tempering period with an organic acidsolution. A 5 log reduction in STEC bacterial load can be seen within1-8 hours at 130° F. Table 16 shows that Salmonella bacterial load canbe reduced by 3 log within 2-8 hours at temperatures of 110° F. andabove, and a 5 log reduction can be achieved within 8 hours at 130° F.

The implementations described above and other implementations are withinthe scope of the following claims. One skilled in the art willappreciate that the present disclosure can be practiced with embodimentsother than those disclosed. The disclosed embodiments are presented forpurposes of illustration and not limitation.

What is claimed is:
 1. A method of reducing bacterial load on grain, themethod comprising incubating a mixture including the grain and anorganic acid solution for a time and temperature sufficient to provideat least a 2 log reduction in an inoculated grain of the same type,wherein: the organic acid is lactic acid, peracetic acid, acetic acid,citric acid or salicylic acid; the time is less than 24 hours; and thetemperature is at least 70° F.
 2. The method of claim 1, wherein theorganic acid solution is included in the mixture in an amount sufficientto provide an organic acid content of at least about 2 x 10⁻⁵ moles pergram grain and a moisture content to the mixture of from 12% to 20%. 3.The method of claim 1, wherein the organic acid solution has aconcentration of organic acid of from 1.5% to 15%.
 4. The method ofclaim 1, wherein the method further comprises a step of preheating thegrain prior to incubation with the organic acid solution.
 5. The methodof claim 1, wherein the time is at least 1 hour and the temperature isat least 100° F.
 6. The method of claim 1, wherein the time is at least1 hour and the temperature is from 110° to about 170° F.
 7. The methodof claim 1, wherein the organic acid is lactic acid.
 8. The method ofclaim 1, wherein the method is performed during tempering of the grain.9. The method of claim 1, wherein the time is less than 12 hours. 10.The method of claim 1, wherein the bacterial load includes Shiga toxinproducing E. coli (STEC).
 11. The method of claim 1, wherein thetemperature is about 70° F.